Results
The Tshr gene of the common vole is 113,629 bp long and consists of 8 introns and 9 exons (Fig. 2A). In this study we sequenced a ~1700 bp region around the beginning of exon 9, comprising 829 bp of intronic and 849 bp of exonic sequences (Fig. 2B). The predicted TSHR protein structure, based on the Microtus arvalis genome (NCBI: txid47230, GCA_007455615.1), comprises seven leucine-rich repeats, a hinge region and seven transmembrane domains (Fig. 2C, D).
Forty-nine single nucleotide polymorphisms (SNPs) were detected (Table S4, S5). Twenty-two intronic SNPs and 27 exonic SNPs were found, from which 23 were synonymous. These SNPs were used to calculate the genetic differentiation of the sampled populations. Pairwise multilocus FST estimates ranged from FST = 0.000 to 0.978 (mean FST = 0.366, with 0 denoting no difference and 1 referring to completely different populations), and reveal high genetic differentiation between the populations sampled (Fig. 3). The high FST values in this species are in agreement with previous studies(Heckel, Burri, Fink, Desmet, & Excoffier, 2005; Martínková et al., 2013). The structure of the observed population differentiation suggests that not only population subdivision but also natural selection may be an explanatory factor. Therefore, it was tested whether the observed SNPs are associated with geographical location. Genetic differentiation between Eastern and Western European populations was larger (FST = 0.247 to 0.978, mean: 0.640) than differentiation among Western European populations (FST= 0.016 to 0.467, mean: 0.234) and among Eastern European populations (FST = 0.000 to 0.648, mean: 0.132), and strongly depends on geographical distance and longitude (Figs. 3, S2, Table S5). The constructed distance tree based on Tshr haplotypes (Fig. S2) together with pairwise multilocus FST analysis (Fig. 3), confirmed that the Western and Eastern European populations belong to different genetic lineages. Given the genetic separation of these lineages, it is appropriate to analyze Western and Eastern European samples separately. To reveal possible patterns of association between SNPs, heatmaps of pairwise linkage disequilibrium (LD) measurements were generated (Fig. S3).